CN1233654C

CN1233654C - Method for renaturing hydroxyapatite and purifying gene recombinant protein simultaneously

Info

Publication number: CN1233654C
Application number: CN 200410022474
Authority: CN
Inventors: 姚少华; 张伶俐; 张兴栋; 范红松
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2004-05-10
Filing date: 2004-05-10
Publication date: 2005-12-28
Anticipated expiration: 2024-05-10
Also published as: CN1569888A

Abstract

The present invention relates to a method for renaturing hydroxyapatite and simultaneously purifying gene recombinant protein. The method is characterized in that hydroxyapatite which has strong adsorption capacity to denatured protein and weak adsorption capacity to a denaturing agent and a reducing agent is used as filling materials; the denaturing agent with high concentration prevents the deposition of the protein on pipelines and column heads. A proper elution method is used for separating protein, the denaturing agent and the reducing agent. The method realizes the folding renaturation of protein, simultaneously separates interest protein and hybrid protein, and improves active recovery rates. The purity of the interest protein reaches 90%.

Description

The method of hydroxyapatite renaturation and while purified genes recombinant protein

One, technical field

The present invention relates to the method for a kind of hydroxyapatite renaturation and while purified genes recombinant protein, belong to the downstream techn of gene engineering field.

Two, background technology

With intestinal bacteria is the gene recombinant protein that the host expresses, and often the overwhelming majority exists with soluble and the active inclusion body form of lifeless matter.Denaturing agent commonly used dissolves inclusion body, removes denaturing agent or reduces its concentration by the method for dialysis or dilution then, and under the condition of no denaturing agent or lower concentration denaturing agent, protein could obtain native conformation, obtains biological activity, and this process is called renaturation.But adopt this two kinds of methods, most of protein will form precipitation, has only small portion protein, and is about 5～20%, obtains renaturation.

According to Liu Guoquan chief editor " biotechnology downstream technology ", Chemical Industry Press's second edition in 2003 adopts liquid chromatography technology recombinant protein matter that good effect is arranged, and activity recovery has greatly improved, and the effect of purifying is arranged simultaneously.Can be used in protein renaturation and simultaneously purifying gel filtration chromatography, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography arranged.Gel filtration chromatography is to utilize protein molecule different with the denaturing agent bulk of molecule, and gradually both is separated, and realizes that renaturation, its key are to form good denaturing agent concentration gradient in the post bed, make albumen break away from denaturing agent gradually; Back three belongs to the adsorptivity chromatogram, all is to utilize the stationary phase of post bed and absorption of proteins reactive force to be better than the reactive force of stationary phase and denaturing agent, and protein is separated with denaturing agent, and this adsorption also can help protein renaturation and prevent albumen precipitation.But, its weak point is all arranged.In the gel filtration chromatography, the concentration gradient of denaturing agent is wayward, and protein precipitates in chromatographic column easily and stops up pillar, and loading protein concentration can not be too high; The purifying ability is relatively poor.At ion-exchange chromatography, the most frequently used denaturing agent Guanidinium hydrochloride also can keep in stationary phase, not only influence column capacity, and often in elution process, flow out chromatographic column, thereby the use of the proteinic denaturing agent Guanidinium hydrochloride of the most effective extraction is restricted with protein.Chinese patent ZL92102727 in the hydrophobic chromatography renaturation, add the salt of high density in sex change liquid, limited protein concentration, and produces precipitation at the column cap place easily.Affinity chromatography only has affinity interaction, narrow application range to a kind of or a few protein.Therefore, adopting good chromatograph packing material, explore suitable operational condition, in the renaturation protein purification, prevent sedimentary generation, is the key issue of liquid chromatography renaturation gene recombinant protein.

Three, summary of the invention

The objective of the invention is to provide the method for a kind of hydroxyapatite renaturation and while purified genes recombinant protein at the deficiencies in the prior art, be characterized in adopting hydroxyapatite as liquid chromatography stuffing, before and after loading, metaprotein adds denaturing agent, the high density metaprotein is carried out renaturation, avoided producing in the protein renaturation process precipitation, improved activity recovery, the purity of target protein reaches 90%.

Purpose of the present invention is realized that by following technical measures wherein raw materials used umber is mole number except that specified otherwise.

The method of hydroxyapatite renaturation and while purified genes recombinant protein:

1. select for use to metaprotein strong adsorptive power is arranged and to the more weak hydroxyapatite of the adsorptive power of denaturing agent and reductive agent as filler, with high density denaturing agent 4～8mol/L urea, prevent the precipitation of albumen at pipeline and column cap place.With elution process albumen is separated with reductive agent with denaturing agent, realize proteic folding renaturation; Simultaneously target protein is separated with foreign protein, realize the purifying of target protein.

2. wash-out denaturing agent, reductive agent

Denaturing agent commonly used is that Guanidinium hydrochloride and urea, reductive agent are dithiothreitol (DTT), with the hydroxyapatite bonding force a little less than, available deionized water or low concentration salt solution are as the Na that contains of 0～0.05mol/L ⁺, NH ₄ ⁺, K ⁺, Ca ²⁺, Mg ²⁺Etc. cationic hydrochloride, vitriol, phosphoric acid salt, Citrate trianion, acetate solution with its wash-out.

3. elute protein

Hydroxyapatite is similar to ion-exchanger to proteic adsorption, and the higher salt of the concentration of available pH＞5 contains Na as 0.1～3mol/L ⁺, NH ₄ ⁺, K ⁺, Ca ²⁺, Mg ²⁺Etc. cationic hydrochloride, vitriol, phosphoric acid salt, Citrate trianion, acetate solution, linear gradient or stagewise gradient be wash-out foreign protein and target protein respectively.

4. pillar regeneration

Behind the target protein wash-out, with the sodium hydroxide of above-mentioned high salt concentration or 0.5～1mol/L with the complete wash-out of all foreign proteins, and then with deionized water or low ion concns salts solution balance pillar, at least 5 column volumes.

The present invention has following advantage:

(1) on metaprotein, adds before and after the sample and use denaturing agent, avoided protein to produce gathering and precipitate;

(2) albumen applied widely, that hydroxyapatite can the combined belt positive and negative charge also can be used for the renaturation and the purifying of several genes recombinant protein in conjunction with uncharged neutral protein; And the albumen of Guanidinium hydrochloride and urea sex change is upper prop directly;

(3) hydroxyapatite is very stable, the experimental result good reproducibility; Filler economy helps industry to amplify.Be applicable to the renaturation and the purifying of genetically engineered downstream recombinant protein.

Four, description of drawings

Fig. 1 is a chromatographic run device synoptic diagram:

1. deionized water or low concentration salt solution groove 2. high density urea solution grooves 3. metaprotein grooves 4. high salt concentration solution tanks 5. pumps 6. hydroxylapatite chromatography posts 7. detectors 8. collectors.

Fig. 2 is last sample synoptic diagram:

9. high density urea 10. metaprotein solution.

Fig. 3 is the renaturation of recombinant human bone morphogenesis protein-2 and color atlas and the electrophorogram in the while purification process:

A, B, C are the foreign protein component, and D is the target protein component, and a, b, c, d are the electrophoretogram of foreign protein component, and e, f are the electrophoretogram of target protein component, and g is a protein molecular weight standard.

Five, embodiment

Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.

Embodiment:

1, recombinant human bone morphogenesis protein-2

With the bacterium inclusion body dissolving that reduces denaturation of will recombinating of Guanidinium hydrochloride and dithiothreitol (DTT), make total protein concentration 25～30mg/ml, concentration of guanidine hydrochloride 6mol/L, the pool alcohol 0.2mol/L of two sulphur Soviet Union.Last batten spare: with 1ml 8mol/L urea balance column cap, the pillar specification is φ 1.6cm * 3cm earlier, and last sex change liquid 1.5ml then, pours into 1ml 8mol/L urea again.Drip washing condition: earlier with deionized water wash-out denaturing agent and reductive agent, again with gradient sodium phosphate difference wash-out foreign protein and target protein.Chromatogram and electrophoresis test result see for details shown in Figure 3, and A, B, C are the foreign protein peak, and a band is the component at A peak, b, c are C peak component, and d is a B peak component, and the g band is a protein molecular weight standard, the D peak is the target protein peak, and e, f are D peak component, and target protein purity is 90%.With direct oxidation after the component desalination of D peak.Oxidizing condition is pH=8.5, reduced glutathion 2mmol/L, Sleep-promoting factor B 1mmol/L.Induce rat embryo myoblast alkaline phosphatase expression of enzymes experiment confirm biological activity, target protein activity recovery about 30～50%.

2, egg albumen N,O-Diacetylmuramidase

With 8mol/L urea and the 0.2mol/L dithiothreitol (DTT) N,O-Diacetylmuramidase that reduces denaturation, the about 35mg/ml of lysozyme concentration, fully sex change is after 4 hours, add biphosphate sodium to final concentration 0.02mol/L, then go up sample 0.2ml in the good hydroxyapatite pillar φ 1.6cm * 2cm of 0.02mol/L biphosphate sodium balance, add the solution that contains 8mol/L urea and 0.02mol/L SODIUM PHOSPHATE, MONOBASIC with 0.5ml before and after the application of sample.Use 0.02M SODIUM PHOSPHATE, MONOBASIC and the drip washing of 0.2M Sodium phosphate dibasic successively, obtain two peaks.With first peak desalination, oxidation, oxidizing condition is: 0.05mol/L Tutofusin tris-hydrochloric acid pH=8.5,2mmol/L reduced glutathion and the oxidation of 0.2mmol/L Sleep-promoting factor B; Second peak directly add reduced glutathion to 2mmol/L, Sleep-promoting factor B to the 0.2mmol/L oxidation.Activity recovery is greater than 85%.

3, porcine pepsin

Denaturation method and the same N,O-Diacetylmuramidase of pillar specification, the about 33.33mg/ml of protein concentration, last sample 0.3ml, front and back 0.5ml8mol/L urea solution.Use deionized water, the drip washing of 0.15mol/L Sodium phosphate dibasic successively, obtain two peaks.After the first peak desalination, add Tutofusin tris-hydrochloric acid pH=8.5 to final concentration 0.05mol/L, 2mmol/L reduced glutathion and the oxidation of 0.5mmol/L Sleep-promoting factor B; Second peak directly adds 2mmol/L reduced glutathion, the oxidation of 0.5mmol/L Sleep-promoting factor B.Activity recovery about 75%.

Claims

1. the hydroxyapatite renaturation and the method for purified genes recombinant protein simultaneously is characterized in that:

(1) selecting hydroxyapatite for use is liquid chromatography stuffing;

(2) in the liquid chromatography renaturation process, adding with concentration before and after the loading metaprotein is the urea of 4～8mol/L, prevents that metaprotein from producing precipitation at pipeline and column cap place;

(3) with deionized water or concentration be the Na that contains of 0～0.05mol/L ⁺, NH ₄ ⁺, K ⁺, Mg ²⁺Or Ca ²⁺Cationic hydrochloride, vitriol, phosphoric acid salt, Citrate trianion or acetate solution with denaturing agent and reductive agent wash-out, make the protein folding renaturation;

(4) be 0.1～3mol/L pH value greater than 5 the Na that contains with concentration ⁺, NH ₄ ⁺, K ⁺, Mg ²⁺Or Ca ²⁺Cationic hydrochloride, vitriol, phosphoric acid salt, Citrate trianion or acetate solution, linear gradient or stagewise gradient be wash-out foreign protein and target protein respectively, realizes the purifying of target protein.

2. as the method for claim 1 hydroxyapatite renaturation and while purified genes recombinant protein, it is characterized in that denaturing agent is Guanidinium hydrochloride or urea.

3. as the method for claim 1 hydroxyapatite renaturation and while purified genes recombinant protein, it is characterized in that reductive agent is a dithiothreitol (DTT).